The ongoing accumulation of vast collections of DNA sequence data has catalyzed the development of novel approaches for profiling the expression of genes at the mRNA level. These methods, while extraordinarily powerful, do not provide direct information on changes either in the levels of proteins or their states of modifications. The development of analogous high throughput methods for directly monitoring protein levels, while increasingly desirable for biological investigations in the post-genome era, presents a formidable analytical challenge. Although recent advances in the use of mass spectrometry (MS) in conjunction with protein/DNA-sequence database search-algorithms allows for the identification of proteins with unprecedented speed, it remains difficult to obtain accurate quantitative information concerning the levels of the identified proteins and the levels of site-specific modifications within individual protein molecules. In the absence of appropriate antibodies, quantitation is usually achieved by autoradiography after metabolic radiolabeling, fluorography, or the use of protein stains. These procedures are dependent on complete separation of the proteins of interest by techniques such as high-resolution 2-D electrophoresis. There remains a pressing need for easier, more reliable means to rapidly profile protein levels. We have devised a novel general method for accurately comparing levels of individual proteins present in cell pools that differ in some respect from one another (e.g., the presence of a mutated gene) and for accuractely determining changes in the levels of modifications (e.g., phosphorylation) at specific sites on the individual proteins. The procedure can be applied to mixtures of proteins, obviating the need for complete separation.